Our goal is to understand the role of molecular motions in the generation of force during muscle contraction, focusing on the proposed rotational motions of myosin heads (crossbridges) on the thick filament relative to the actin-containing thin filament. Direct measurements will be made of the orientation, rotational motion, and translational motion of spin-labels and optical probes attached selectively and rigidly to myosin heads, using spectroscopic instrumentation, theory, and methods that we are developing. Following preliminary work on purified myosin and myofibrils, the major emphasis will be on work with glycerinated fibers. Electron paramagnetic resonance (EPR) experiments on spin-label attached to the myosin head in oriented fibers will be performed to study the orientation distribution of heads in various physiological states (rigor, relaxation, isometric tension, etc.). Saturation transfer EPR experiments on the same preparation will provide information about the time-averaged rotational motion of the heads. Using pulsed laser excitation, time-resolved rotational motions of dyes attached to myosin heads will be detected by absorption or emission antisotrophy. Analogous experiments will be performed on actin motions. Finally, fluorescence energy transfer from labeled myosin heads to labeled actin will provide a direct probe of translational motion of myosin heads relative to actin subunits. The results should have a major impact on muscle biophysics, and the spectroscopic methods developed should have much broader applications.